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not been possible. This is preciselywhat. J.J. Kirkland and J.L.Glajch have achieved with the4-solvent, 7-step technique developed in the. DuPont labo...
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A Brief Look at 4-solvent Theory The quality of an HPLC sepa­ ration is judged by its resolution, until now, all advances in chromotographic technique have mainly involved column efficiency, reten­ tion or capacity. Developing a model or rationale for optimum selection of the mobile phase had not been possible. This is precisely what J.J. Kirkland and J.L. Glajch have achieved with the 4-solvent, 7-step technique developed in the Du Pont laboratories. Their first step was to use the Snyder selec­ tivity triangle'". In this triangle, the most com­ mon HPLC solvents have been divided into eight major groups, each of which has different selec­ tivity in a separation. Solvent groups are placed within the trian­ gle on the basis of their relative strength as proton acceptors, pro­ ton donors, or dipole interaction. Those groups closest to the ver­ tices of the triangle are strongest in these factors. (Fig. 1). In making a choice of three solvents for optimizing selectivity, solvents are chosen from groups nearest to the three vertices of the triangle, so as to produce the larg­ est differences in solvent action. Finally, the weak or strengthadjusting solvent—water or hexane—is chosen. Therefore, four solvents are required to carry out the optimization routine. In developing the 4-solvent technique, Kirkland and Glajch use a triangle to define the entire selectivity space of a separation, Fig. 2. This triangle may be plot­ ted within the confines of seven

depending on the mode of the experiment. Solvent compositions for the final four experiments are fixed; the blend ratios are defined on the optimization triangle sides and center, Fig. 2. CH,OH/H,0

CH.OH/CH.CN. Η,Ο /

definitive isocratic experiments using combinations of the four solvents selected. The first of these experiments uses two of the four solvents (methanol-water, for example) which defines one vertex of the optimization triangle. The remain­ ing two vertices are defined by two additional experiments, again using each of the other two sol­ vents, plus water or hexane, Proton Acceptor (Base)

CH,OH/THF/Hp CH.OH CH.CN THF Η,Ο

CH,CN/H 2 0

CHjCN/THF/H 2 0

THF/Η,Ο

Fig. 2. The 7-steps and defined concentrations of the solvents used for each step are shown in this triangle.

Following these seven chro­ matographic runs, an optimum solvent region may be defined from visual examination of the chromatograms. Additional refine­ ments may be made for the sepa­ ration and require one or two additional runs. Detailed theory and explana­ tion of the optimization technique will be found in references below. References 1. L.R. Snyder: J.Chrom. Sci.. /6(1978). 223 ff. 2. J.L Glajch. J.J. Kirkland. KM. Squire. J.M. Minor; J. Chromatography 199 (1980), 57 ff.

Proton Donor (Acid)

"

Dipole Interaction

Fig. 1. The Snyder selectivity triangle divides the most common HPLC solvents into eight groups. Position of the groups within the triangle depends on their rela­ tive strength as proton acceptors, proton donors, or dipole interaction.

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